Figure 1
WFA has osteogenic effect in vitro. (a) Structure of WFA. (b) Effect of WFA on primary osteoblast proliferation. Primary osteoblasts were cultured in increasing concentrations of WFA and Bzb for 24 h and harvested for cell proliferation direct cell count for cell proliferation assay (I) and BrdU incorporation cell proliferation assay (II). The results were expressed as relative cell growth in percentage, which was compared with control group. We set the control group as 100. Values represents mean±S.E. of three independent experiments (n=3). *P<0.05, **P<0.01 when compared with control. (c) WFA treatment of MCOs for 48 h in osteoblast differentiation medium significantly increased ALP production compared with control. Values represent mean±S.E. of three independent experiments (n=3). **P<0.01, ***P<0.001 compared with control vehicle group. (d) Photomicrographs show that treatment of MCOs with WFA in osteoblast differentiation medium significantly increased mineralized nodules compared with control, as assessed by alizarin Red-S staining. (e) Quantitation of mineralization (Alizarin Red-S stain) data are shown as OD at 405 nm. Values represent mean±S.E. of three independent experiments (n=3). ***P<0.001 compared with control vehicle group. (f) WFA (10 nM) treatment of MCOs increased expression of osteogenic genes. Figure shows increased expression of RunX2, OCN and ColI with WFA treatment. Expression was normalized to GAPDH internal control. Values represent mean±S.E. of three independent experiments (n=3). *P<0.05, ***P<0.001 compared with vehicle control group. (g) WFA exerts anti-apoptotic effects in osteoblasts. Using Becton Dickinson FACS and FL-H channel (annexin-V) and FL2-H channel (PI) data show that WFA treatment inhibited apoptosis of osteoblast cells. Shown are representative dot plots. (h) Quantification of flow cytometry data are shown as percent of total cells. Values represents mean ±S.E. of three independent experiments (n=3). *P<0.05 compared with control for early apoptosis and ##P<0.01 compared with control for late apoptosis
